Arizona State University
SOP No.: 2 v 1.0
Effective Date: 3/29/2009
Page 1 of 15
Title: Production and purification of polypeptide libraries
Approved:
Kathryn Sykes
ASU PI
Author: Kathryn Sykes
Date: 6/5/2009
1. Introduction
Francisella tularensis (FTU) is an intracellular pathogen that can be posed as a bioterrorism
agent. The search for vaccine against the FTU pathogen is under research investigation since
the bacteria’s intracellular lifestyle and the mechanism of virulence are not well understood.
Although a live attenuated vaccine (LVS) has been developed and used for years in the USA at
USAMRIID, it does not provide a high quality protection against respiratory related symptoms in
humans. The high throughput protein production system supported by in vitro translation can
provide a complete proteome of the pathogen which can be used for antigen identification and
their evaluation as prophylactic molecular vaccine candidates.
2. Purpose
The purpose of this SOP is to describe a comprehensive scalable protocol for the construction of
FTU Linear Expression Elements (LEEs), their in vitro translation (IVT) into polypeptides, and
polypeptide purification.
3. Responsibility
This SOP will be performed by laboratory scientists. The data and results will be reviewed by
the Principal Investigator.
4. Scope
This protocol can be used for in vitro generating any polypeptide of 100 to 400 amino acids in
length for which corresponding genomic DNA samples are available. This protocol is used in
conjunction with the investigation of developing a tularemia molecular vaccine at the Biodesign
Institute, Center for Innovations in Medicine. The protocol is used in the Biodesign Institute,
CIM, building B, rooms 229 (general laboratory area) and B237A (radioactive material assigned
area).
5. Precautions
Laboratory personal protective equipment (lab coats, gloves, eye goggles etc.) is worn and
ASU’s general laboratory safety regulations are followed and reinforced at all time. In vitro
translation procedure involves using S35 radioactive material. Personnel are properly trained in
handling and disposing radioactive material by a certified ASU radiation safety officer.
6. Materials
6.1. LEE Construction
6.1.1. Supplies
 Linear Prokaryotic Promoter and Terminator with universal adapters
 Primers (Invitrogen 5791 Van Allen Way Carlsbad, CA 92008)
o Gene specific primers with universal adapters,
o Universal Adapter Forward primer (5-Univ-F):
ATAGGCGGAAGCGGATTG
o T7 Thio Forward promoter primer (T7-Thio-F)
GCGAAATTAATACGACTCACTATAGGG
o T7 Thio Promoter Reverse primer (Thio-R):
CAATCCGCTTCCGCCTATGGCCAGGTTAGCGTCGAGG
o T7-Term-His-Univ-F
ACCCAACCTCCCTCCCACCATCATCATCATCATTAATAAAAGGGCG
o T7 Reverse Terminator primer (T7-Term-R) :
ATCCGGATATAGTTCCTCCTTTCAG
 iProof HighFidelity DNA Polymerase (Bio-Rad 1725302)
 DyNazyme II polymerase (Finnzymes F-50BL)
 pEXP5-NT expression vector from Invitrogen.
 pET32b vector from Novagen.
 FTU wild type genomic DNA (from UNM)
 QIAquick Gel extraction kit (Qiagen 28704)
 PCR Plate (Eppendorf Twin.tec PCR Plate 96, skirted 951020486)
 96-well agarose electrophoresis gel, Invitrogen E-gel 2% Agarose, cat# G7008-02
 E-gel Low Range DNA Ladder (12373-031)
 PureLink 96 PCR Purification Kit (Invitrogen K3 100-96)
 UV Half Area Plate 96 Well (Corning 3679)
 dNTP Mix (Promega U1515)
 Agarose (RPI Research Products International Corporation, cat # A20090-500.0)
 100 bp DNA ladder (New England Biolabs N3231L)
 Molecular grade water (Millipore filtered in-house)
6.1.2. Equipment
 Biomek® FX Laboratory Automation Workstation (Beckman Coulter)
 PCR machine: Mastercycler epGradient S (Eppendorf )
 Beckman Coulter Allegra 25R benchtop centrifuge
 NanoDrop (Thermo Fisher Scientific, ND-100)
 Ranin multi channel pipette.
 Power supply for running E-Gel, E-base (Invitrogen, cat# EB-M03)
 Software for editing E-gel image, E-editor (Invitrogen)
 Molecular Imager ChemiDoc XRS System (Bio-Rad 170-8070)
 Plate vacuum manifold (Eppendorf, Perfect Vac Manifold, single Basic, Cat# 0032
007.708)
 SpectraMax 190 (Molecular Devices)
 SpeedVac Concentrator ( Thermo Electron Corporation ISS 110)
6.2. aTrx Antibody Magnetic bead Preparation:
6.2.1. Supplies
 15-mL presterilized centrifuge tubes (VWR cat# 89004-368)
 Mouse Anti Trx-Tag Monoclonal Antibody. Genescript Corp. Cat# A00180
 Dynal beads M-280 Tosylactivated. (Magnetic beads) Invitrogen Cat# 142.04
 Boric Acid (H3BO3) MW 61.83 Fisher Chemicals Cat# A78-500
 Ammonium Sulfate (NH4)2SO4 JT Baker Cat# 0792-05
 Phosphate Buffer Saline (PBS) 1x Sterile 1L Cellegro, cat# 21-040-CM
 Bovine Serum Albumin (BSA) Fraction V Sigma cat# A7906-500G
 Buffer A: 1M Boric Acid, 3M Ammonium Sulfate pH 9.5
 Buffer B: 1X PBS pH 7.4, 0.5% BSA(w/v)
 Buffer C: 1X PBS pH 7.4, 0.1% BSA(w/v)
6.2.2. Equipment:
 RTS ProteoMaster ( Roche cat# 3 265 650)
 Eppendorf MixMate plate shaker
 Plate Magnet: Promega Magna Blot 96 Magnetic Separation Device cat # U8151
 Invitrogen 15ml Dynal MPC-6 magnetic separator, cat# 120-02D
 Rannin 200 ul multi-channel pipette
6.3. In vitro Translation
6.3.1. Supplies:
 PURExpress In Vitro Protein Synthesis Kit (New England Biolabs Cat# E6800S)
 EasyTag™ L-[35S]-Methionine, 5mCi (185MBq), Stabilized Aqueous Solution,
Shipped in Lead (PerkinElmer Life and Analytical Sciences, NEG709A005MC)
 FTU LEE from LEE construction step.
 1.2ml square-well deep-well 96well plates (ABgene AB-1127)
 96-well square-well plate mats (ABgene AB-0675)
 Repeater Pipette Tips
 TCA assay plates: Multiscreen HTS, FC (Millipore Cat# MSFCN6B50)
6.3.2. Equipment
 Repeater pipette
 Ranin multichannel pipette
 Gene Machines HiGro Orbital Incubator
 Beckman Coulter Allegra™ 25R Centrifuge, TS-5.1-500 rotor
 10-μl multi-channel pipette
 Plate vacuum manifold (Eppendorf, Perfect Vac Manifold, single Basic, Cat# 0032
007.708)
 Tri-Carb 2900TR liquid scintillation counter (PerkinElmer A290000)
 Gel box (Criterion dodeca cell Bio-Rad)
 Gel Dryer (Bio-Rad Model 583)
 Storage Phosphor Screen (Amersham Biosciences)
 Typhoon TRIO+ variable mode imager (Amersham Biosciences)
6.4. Quality control:
6.4.1. Supplies
 TCA Assay Plates (Millipore, Multiscreen HTS, FC cat# MSFCN6B50)
 Glass Rod
 10% TCA (VWR cat#VW3372-2)
 95% Ethanol
 6ml Scintillation Vial (Research Products International Corp. #125514)
 Scintillation Fluid (Ultima Gold PerkinElmer)
 4X gel loading buffer (Bio-Rad cat#161-0791)
 10X gel loading reducing agent (Bio-Rad cat#161-0792)
 26-well Criterion XT Bis-Tris Gel, 4-12%, 1mm (Bio-Rad cat#345-0125)
 Precision Plus protein Kaleidoscope molecular marker (Bio-Rad cat#161-0375)
 Gel running buffer (Criterion XT, MES Bio-Rad cat#161-0789)
 Gel fixation solution (40% methanol, 10% acetic acid)
 Storage Phosphor Screen (Amersham Biosciences)
6.4.2. Equipment:
 Repeater pipette
 Ranin multichanel pipette
 Gene Machines HiGro Orbital Incubator
 10-μl multi-channel pipette
 Plate vacuum manifold (Eppendorf, Perfect Vac Manifold, single Basic, Cat# 0032
007.708)
 Tri-Carb 2900TR liquid scintillation counter (PerkinElmer A290000)
 Gel box (Criterion dodeca cell Bio-Rad)
 Gel Dryer (Bio-Rad Model 583)
 Typhoon TRIO+ variable mode imager (Amersham Biosciences)
7. Procedures
7.1. LEE Construction
7.1.1. Preparation of Promoter and Terminator with N-Terminal Thioredoxin fusion and Cterminal His tag at ASU, prepared in sufficient bulk for use throughout experiment).
7.1.1.1.
PCR Amplification of Promoter is from pET32b vector which contains
Thioredoxin (Trx) at the N-terminus. Terminator is from pEXP5-NT vector. The
forward primer contains the His-tag sequence. PCR conditions for amplifying the
promoter and terminator are similar. The primers for each are as follows:
 Promoter :
o Forward Primer = T7-Thio-F
(GCGAAATTAATACGACTCACTATAGGG)
o Reverse Primer = Thio-R
(CAATCCGCTTCCGCCTATGGCCAGGTTAGCGTCGAGG)
 Terminator:
o Forward Primer= T7-Term-His-Univ-F
(ACCCAACCTCCCTCCCACCATCATCATCATCATTAATAAAAGG
GCG)
o Reverse Primer= T7-Term-R (ATCCGGATATAGTTCCTCCTTTCAG)
7.1.1.2.The PCR conditions are as follows:
Table 1. Description of amplification conditions used for FTU ORF production
1
2
3
4
5
6
7
PCR Master Mix
H2O
iproof 5x
dNTP (10 mM)
F Prim (10uM)
R Prim (10uM)
Templ (5 ng/μl)
Iproof Poly
Total
No. of Rxn
1X
MM1
36.5
10
1
0.5
0.5
1
0.5
50
3854.4
1056
105.6
52.8
52.8
105.6
52.8
5280
1
96
PCR Cycler Protocol
Temp (OC)
Time (Min)
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Step 7
98
2
98
0.75
50
0.5
72
1
34X Goto step 2
72
5
4
Hold
7.1.1.3. Run the entire reaction on 1.2% agarose gel with Ethidium bromide at 200V for 30
minutes in 1/2X TBE buffer ( 0.05M Tris, 0.05M Boric acid, 1mM EDTA)
7.1.1.4. Image the gel on Chemidoc imager.
7.1.1.5. Cut the appropriate size band from the gel using a new razor blade.
7.1.1.6. Weigh the gel slice on a balance in a pre-scaled 15 ml conical tube approximated of
7.065 gram.
7.1.1.7. Follow the Qiagen gel purification kit protocol for purifying the Promoter.
7.1.1.8. Quantitate the yield by Nanodrop using absorbance at 260nm. See Section 10 for
calculations.
7.1.1.9. Dilute to 0.05ng/μl, aliquot 105μl to 0.5 polypropylene micro centrifuge tube, and
store at -20°C.
7.1.2. FTU ORF production and Overlapping PCR for LEE production
7.1.2.1. Note: The master mix and the PCR conditions are explained in the tables 2 to 6
Schu S4 DNA was provided by the University of New Mexico. UNM tested the
SCHU S4 DNA for sterility by lack of CFU, prior to shipping to ASU.
7.1.2.2. Mix the gene specific forward and reverse FTU primers for the individual ORFs
and dilute to 0.2μM concentration using the FX Robot. An Excel file loads into
computer script of the FX robot so that all genes are tracked. The computer script
on FX Robot directs the pipetting.
7.1.2.3. Transfer 12.5μl of this PCR primer mix to 96-well PCR plate using multi-channel
pipette.
7.1.2.4. Into each well of the 96-well PCR plate containing primer mix, add 12.5μl of FTU
ORF PCR master mix prepared as shown in table 2.
7.1.2.5. Carry out the PCR in Eppendorf PCR machine according to conditions in the table
6
7.1.2.6. Add 5μl of the FTU ORF PCR from step 7.1.2.5 to another 96-well PCR plate, then
add 20μl of the master mix for Assembly Overlap step 1 (as shown in table 3) for
each reaction in the plate.
7.1.2.7. Carry out the second PCR in Eppendorf PCR machine according to Assembly
overlap PCR conditions as shown in the table 6.
7.1.2.8. Add 5μl of the Assembly Overlap PCR step 1 from step 7.1.2.7 to another 96 well
PCR plate, and then add 20μl of master mix for Assembly Overlap PCR step 2
prepared as shown in table 4.
7.1.2.9. Carry out the third PCR in Eppendorf PCR machine according to conditions in the
table 6.
7.1.2.10. Add 5ul of the Assembly Overlap PCR step 2 from step 7.1.2.9 to another 96 well
PCR plate, and then add 95μl of the master mix for Assembly overlap PCR
amplification prepared as shown in table 5.
7.1.2.11. Carryout the fourth PCR in Eppendorf PCR machine according to conditions shown
in table 6:
7.1.3. LEE construction Quality Control
7.1.3.1. Run10 μl of the PCR reaction from step 7.1.2.11 on 96 well E-Gel of Invitrogen
using the protocol specified by the company.
7.1.3.2. Image the gel on the Chemidoc.
7.1.3.3. Analyze the image using E-editor of Invitrogen.
7.1.3.4. Store the image and results on the GEMS database.
Table 2: PCR amplification of ORF from FTU genomic DNA
ORF PCR
1
2
3
4
5
6
1X
4.25
5
0.5
12.5
2.5
0.25
25
1
H2O
iproof 5x buffer
dNTP (10 mM)
Sp F + R Primer Mix (0.8uM)
Wt DNA
iProof Pol
Total
No. of Rxn
HTP MM1
1481.6
1743.0
174.3
4357.5
871.5
87.2
8715.0
332.0
Table 3: PCR overlapping of terminator to FTU ORF from Step 1
Assembly PCR step 1
1
2
3
4
5
6
7
8
H2O
iproof 5x
dNTP (10 mM)
Univ F Primer(10uM)
T7 Term R Primer (10uM)
Terminator (0.05ng/ul)
ORF (Wt PCR)
iProof Pol
Total
No. of Rxn
1X
12.75
5
0.5
0.25
0.25
1
5
0.25
25
1
HTP MM1
4444.7
1743.0
174.3
87.2
87.2
348.6
1743.0
87.2
8715.0
332.0
Table 4: PCR overlapping of promoter for LEE generation
Assembly PCR step 2
1
2
3
4
5
7
8
10
H2O
iproof 5x
dNTP (10 mM)
T7-Thio-F Prim (10uM)
T7 Terminator R Primer (10uM)
Promoter (0.05ng/ul)
ORF + Term PCR
iProof Pol
Total
No. of Rxn
1X
12.75
5
0.5
0.25
0.25
1
5
0.25
HTP MM1
4444.7
1743.0
174.3
87.2
87.2
348.6
1743.0
87.2
25
1
8715.0
332.0
Table 5: PCR amplification of LEE template from Overlap PCR step 2
LEE Amplification
1
2
3
4
5
6
7
1X
80
10
2
1
1
5
1
100
1
H2O
Dnazyme 10x
dNTP (10 mM)
T7-Thio-F Primer (10uM)
T7 Terminator R Primer (10uM)
Template (5ng/ul)
Dynazyme Poly
Total
No. of Rxn
HTP MM1
27091.2
3386.4
677.3
338.6
338.6
1693.2
338.6
33864.0
332.0
Table 6: Thermal cycler programs for ORF, Assembly by overlapping PCR (Steps 1 and 2)
and LEE generation
Assembly PCR (Step
1&2)
ORF PCR
Step in
PCR
program
0
Temp (
C)
Time
(sec)
0
Temp (
C)
Time
(sec)
LEE Amplification
Temp
0
( C)
Time (sec)
1
98
30
98
120
98
120
2
98
10
98
45
98
45
3
50
30
55
30
55
30
4
72
15
72
60
72
5
4X Go to step 2
6
98
10
72
300
72
300
7
62
30
4
Hold
4
Hold
8
72
15
9
19X Goto step 6
10
72
60
11
4
Hold
19X Go to step 2
60
14X Go to step 2
7.1.4. PCR Cleanup
7.1.4.1. Remove primers and dNTPs in the overlap PCR amplification reaction from step
7.1.2.15 using the PureLink 96 PCR Purification Kit, according to the specifications
of the company.
7.1.4.2. Elute overlap PCR product with 100ul of Molecular grade RNase and DNase free
water, directly into the half volume Corning UV plate. This should yield 83-85ul of
final purified PCR products.
7.1.5. DNA Quantification
7.1.5.1. The half volume Corning UV plate can be used for the 96-well format
spectrophotometer (SpectraMax 190). 83-85ul should be sufficient volume to get
good absorbance. It is necessary for the spectrophotometer to have at least 50ul of
volume for reliable reading.
7.1.5.2. Softmaxpro V5 is used as the program to operate the SpectraMax 190 instrument.
Specifically, to collect the data and calculate the yield, preset protocol of DNA
RNA & PathCheck is used with the following alterations.
7.1.5.2.1. Under Plate then under Setting and then under Pathcheck- unselect the plate
background constants.
7.1.5.2.2. Under the Plate then under the Template: Assign cells H10, H11 and H12 as
blank (83ul water is used as blank). Assign the rest as Group 01 unknowns.
7.1.5.2.3. Under the Samples7.1.5.2.3.1. Assign the Concentration Factor as 38.1
7.1.5.2.3.2. Create a new column with heading total μg DNA, and the formula is
“Conc*83”
7.1.5.2.4. A template of the protocol with the alterations can be made for ease of use.
7.1.5.3. The Softmaxpro can generate and text file for the table of calculations.
7.1.5.4. Ensure that all of the samples have DNA amount of at least 0.5μg. If the amount of
any of the samples is lower, repeat the amplification reaction in step 7.1.2.11 and
carry out individually PCR clean up as in step 7.1.4.
7.1.6. DNA Preparation for IVT
7.1.6.1. Transfer 250ng of DNA template each well to Thermo Scientific 1.2 ml 96 Deepwell plates.
7.1.6.2. Evaporate the liquid completely using speed vacuum. Plates can be stored at
-20oC and it should be used as soon as possible.
7.2. aTrx-Tag Magnetic Beads generation:
7.2.1. Buffers
7.2.1.1. Buffer A: dissolve 6.18g Boric Acid (H3BO3), 31.71g Ammonium Sulfate
(NH4)2SO4 in 95 ml of ultra pure water and adjust the pH to 9.5 with NaOH tablets,
bring the final volume to 100ml with ultra pure water.
7.2.1.2.Buffer B: Dissolve 0.5g of BSA fraction V, in 100ml of 1X PBS.
7.2.1.3.Buffer C: Dissolve 0.1g of BSA fraction V, in 100ml of 1X PBS.
7.2.2. Bead Preparation.
7.2.2.1.Use the ratio of 25ul beads per reaction to calculate the amount of beads to be
prepared. (Following example will be for 100 reactions)
7.2.2.2.The beads settle pretty fast, therefore it is very important to mix the magnetic beads
stock very thoroughly by pipetting up and down. After mixing quickly pipette
appropriate amount of beads into low protein binding 15ml tube. (2.5ml for 100rxn)
7.2.2.3.Place the tube on the magnet for 2 minutes.
7.2.2.4.The magnetic beads should have travelled to the wall adjacent to the magnet and stay
there. If the solution seems cloudy and still has beads floating, wail until the liquid
clears. Pipette the liquid out and discard it.
7.2.2.5.Quickly add the same volume of 0.5X Buffer A. (2.5ml for 100rxn)
7.2.2.6.Take the tube out of the magnet.
7.2.2.7.Mix the beads thoroughly to suspend all of the beads.
7.2.2.8.Place the tube on the magnet for 2 min.
7.2.2.9.The magnetic beads should have travelled to the wall adjacent to the magnet and stay
there. If the solution seems cloudy and still has beads floating, wail until the liquid
clears. Pipette the liquid out and discard it.
7.2.2.10. Repeat steps 7.2.2.5 to 7.2.2.9
7.2.2.11. The beads are now washed and ready for the antibody. Do not wait more than 1
minute before adding the Antibody.
7.2.3. Anti-Trx tag antibody binding to beads.
7.2.3.1. Use the ratio 3ug anti-thioredoxin antibody to 5ul starting volume of beads. Be sure
that the antibody is in buffer that does not contain an amine group. PBS is the tested
buffer for this protocol
7.2.3.2.Add an appropriate volume of anti-thioredoxin antibody to the washed beads from
step 7.2.2.11. (If anti-thioredoxin antibody comes in 1ug/ul concentration. For the
example above, 100 reactions, we used 2.5 ml beads, therefore we will add 1.5ml of
anti-thioredoxin antibody)
7.2.3.3.Immediately add the same volume of Buffer A as the volume of the antibody. (1.5ml
above example)
7.2.3.4.Mix well and suspend all of the beads.
7.2.3.5.Set the Thermo mixer R to 37 OC, and 1100 rpm
7.2.3.6.Place the suspended beads with antibodies in the incubator and incubate with shaking
for 24 hours.
7.2.4. Blocking and preparation of beads for IVT
7.2.4.1.After 24-hour incubation with antibody, the antibody should have bound to the
magnetic beads. Place the tube on the magnet and allow the beads to separate for
2min. Be sure the solution has become clear.
7.2.4.2.Pipette out the antibody solution.
7.2.4.3.Add the same volume of Buffer B (3ml for above example) to the beads and incubate
on the Thermo mixer R at 37 OC, and 1100 rpm for 1 hour.
7.2.4.4.Place the tube on magnet and allow the beads to separate for 2min. Be sure the
solution is clear.
7.2.4.5.Pipette out Buffer B, and discard.
7.2.4.6.Add the same volume of Buffer C (3ml for above example). Take tube off of the
magnet and suspend the beads completely.
7.2.4.7.Place the tube on magnet and allow the beads to separate for 2 minutes. Be sure the
solution is clear.
7.2.4.8.Pipette out Buffer C, and discard.
7.2.4.9.Repeat steps 7.2.4.6 to 7.2.4.8
7.2.4.10. Add the same volume of Buffer C as the original volume of beads taken. (2.5ml
for 100rxn)
7.2.4.11. The beads are now ready for the IVT reaction. Store the anti-thioredoxin
antibody magnetic beads at 4 OC. Use these beads as early as possible. If these beads
are to be used after 2 weeks, test their performance before use. Each reaction will get
5ul of beads.
7.3. Bacterial, Coupled In vitro Transcription and Translation
7.3.1. Use the PURExpress In Vitro Protein Synthesis Kit from New England Biolabs for
making the FTU proteins in vitro. For the most part the protocol specified by the company
is used.
7.3.2. Add 7.0 ul of RNase DNase free molecular grade water to each well in IVT reaction plate
and the QC plate containing dried DNA LEE template.
7.3.3. Briefly spin the plate in swing bucket centrifuge at centrifuge at 402 x g for 2 minutes to
bring the water to the bottom of the well.
7.3.4. Allow the DNA to dissolve over night at 4 OC. Once dissolved, use the plate within 24
hours.
7.3.5. IVT Master Mix: Prepare the IVT master mix as follows.
7.3.5.1.Turn on the Gene Machines Higro incubator, and set the temperature to 37 OC
7.3.5.2.Add the appropriate volume of beads with anti-Trx antibody prepared in section 7.2.
to a 15 mL centrifuge tube.
7.3.5.3.Place the tube on magnet. Allow 2 minutes for the beads to adhere to the magnet.
7.3.5.4.The magnetic beads should have travelled to the wall adjacent to the magnet and stay
there. If the solution seems cloudy and still has beads floating, wail until the liquid
clears. Pipette the liquid out and discard it.
7.3.5.5.Immediately add 12.5 μl of Solution A and 5μL of solution B from NEB PURExpress
In vitro Protein Synthesis kit per reaction to the tube.
7.3.5.6.Mix well by pipetting up and down gently. Be sure to suspend the beads thoroughly.
Do not mix too roughly to avoid foaming.
7.3.5.7.Aliquot out appropriate amount of IVT Master mix for the QC Plate. 17ul per
reaction. Each plate will have 12 reactions run as QC.
7.3.5.8.Add appropriate volume of 35S labeled methionine to the master mix; approximately
3.3 μCi per a QC reaction. This will be referred to as Hot IVT Master mix.
7.3.5.9.Add 17.5ul of IVT master mix to each well of dissolved DNA template plate. Take
care to keep the beads well suspended while distributing to ensure that each well
receives the same amount of beads.
7.3.5.10. Similarly add 18ul of Hot IVT Master mix to the QC plate with dissolved DNA
Template.
7.3.5.11. Place the seal mat on the plate and secure it well.
7.3.5.12. Place the IVT reaction plates in the Higro incubator.
7.3.5.13. Shake at 650 rpm for 1 hour at 37OC.
7.3.6. An example of calculation for 1 plate of IVT reaction is given below. Here 82 proteins
are made in one plate. Another set of FTU LEE and GFP templates (total of 13 reactions)
are placed into another separate 96-well plate due to radioactive 35S labeled methionine
added for TCA counts and PAGE gel for quality control.
Table 7. Calculation of polypeptide yields
HTP FTU IVT protein
production
10/20/2008
Antibody Calculator
Rxns
1
96
0
1 Meg Beads
25
2640
0
2 aTrx Antibody
15
1584
0
3 Buffer A
15
1584
0
Plate Preparation
Add 7.0 ul of Rnase Free H2O to ea well to
dissolve the DNA template.
IVT set up Master Mix
1 aTrx Beads
25
2640
Remove the Supernatant
2 Soln A
12.5
1320
3 Soln B
5
528
Add 17.5ul of IVT Master Mix to ea well
0
0
0
Hot Master Mix
1
12
0
1 IVT MMX
17
214.2
0
2 35S Met
1
12.6
0
Add 18ul Hot IVT MMX to ea QA Well
7.4. Anti Trx-tag magnetic beads purification
7.4.1. As the IVT reaction proceeds, the newly synthesized protein gets captured by the aTrxtag beads.
7.4.2. Once the IVT reaction is complete. Place the plate on to the plate magnet. (Promega
Cat# )
7.4.2.1.Allow the beads to migrate to the magnet for 2 minutes.
7.4.2.2.The magnetic beads should have travelled to the wall adjacent to the magnet and stay
there. If the solution seems cloudy and still has beads floating, wait until the liquid
clears.
7.4.2.3.Pipette the liquid out and save it in a fresh clean plate.
7.4.2.4.Immediately proceed to wash step. Do not allow the beads to dry.
7.4.3. Wash of protein-bound magnetic beads:
7.4.3.1.Add 100ul of PBS, pH 7.4
7.4.3.2.Mix well by pipetting up and down gently to suspend all of the beads.
7.4.3.3.Place the plate on the magnet for 2 minutes.
7.4.3.4.The magnetic beads should have travelled to the wall adjacent to the magnet and stay
there. If the solution seems cloudy and still has beads floating, wait until the liquid
clears. Pipette the liquid out and discard it.
7.4.3.5.Repeat steps 7.4.3.1 to 7.4.3.4 two more times.
7.4.4. For final storage before shipment. Add 100ul of 1X PBS to each well and store at -20oC.
8. Quality control
8.1. Sample preparation:
8.1.1. Follow the bead washing procedure in 7.4.1 to 7.4.3 for the QC plate. This work should
be done in designated area for using radioactive material.
8.1.2. Add 20 μl of loading buffer with 5% beta-mercaptoethanol final.
8.1.3. Heat the samples at 95OC for 10min.
8.1.4. Mix well by pipetting up and down or on Eppendorf Mix mate plate shaker at 1000rpm
for 1 minute.
8.1.5. Place the plate on to the plate magnet. (Promega Cat# V8151 )
8.1.6. Allow the beads to migrate to the magnet for 2min.
8.1.7. The protein should have released from the beads and be dissolved into the loading buffer.
8.2. TCA Assay: quantitation of de novo polypeptide yield
8.2.1. Spot 5ul of loading buffer directly onto glass fiber filter of TCA Assay plate from each
well using multichannel pipette. Allow 15-minutes to air dry.
8.2.2. Place the TCA Assay Plate onto the Eppendorf plate vacuum manifold as directed by the
manufacture.
8.2.3. Pipette 200ul of 10% TCA into each well with multichannel pipette, and using aerosol
barrier tips.
8.2.4. Incubate for 5min.
8.2.5. Turn the vacuum on and allow all of the TCA to slowly pass through the filter.
8.2.6. Turn the vacuum off.
8.2.7. Pipette 200ul of 10% TCA into each well with multichannel pipette, and using aerosol
barrier tips.
8.2.8. Turn the vacuum on and allow all of the TCA to slowly pass through the filter.
8.2.9. Turn the vacuum off.
8.2.10. Repeat steps 8.1.7 to 8.1.9 two times.
8.2.11. Pipette 200ul of 95% ethanol into each well with multichannel pipette, and using aerosol
barrier tips.
8.2.12. Turn the vacuum on and allow all of the ethanol to slowly pass through the filter.
8.2.13. Turn the vacuum off.
8.2.14. Repeat steps 8.1.11 to 8.1.13 two times.
8.2.15. With the Vacuum on, allow the filters to dry for 5min.
8.2.16. Stop the vacuum. Take the plate off the manifold. Remove excess liquid from the bottom
of the plate by, forcefully tapping the plate on a stack of paper towels having the bottom
face down. Repeat this as many times as needed to dislodge the excess liquid.
8.2.17. Place plate back on the vacuum manifold. Turn the vacuum on and allow the filter to dry
for 5 min.
8.2.18. Once dry, transfer the Glass filter from plate to scintillation vial as follows.
8.2.19. Hold the plate such that a well is directly above the scintillation vial.
8.2.20. Push the Glass filter through the well from the top with a glass rod directly into a
scintillation vial. Be sure that the support membrane below the glass fiber filter is
detached from the glass fiber filter.
8.2.21. Repeat 8.1.16 and 8.1.17 for the entire plate.
8.2.22. Place enough scintillation fluid in each scintillation vial with glass filter to complete
cover the filter. The filter should be soaked with the scintillation fluid, and become
transparent. (Approximate 1ml of Ultima Gold scintillation fluid).
8.2.23. Place the vial into scintillation counter and record CPMs for each sample.
8.2.24. Calculate the total ug of protein produced from CPMs using the formula provided in the
PURExpress™ In Vitro Protein Synthesis kit user manual page 10.
8.3. Gel Electrophoresis: Confirming molecular weight of radioactive labeled synthesized proteins
8.3.1. Prepare Criterion electrophoresis unit as specified by the company, using 24-well
Criterion XT Bis-Tris Gel, 4-12%, 1mm, Gel Electrophoresis Buffer (Criterion XT, MES
Bio-Rad) in the Gel Electrophoresis box (Criterion dodeca cell Bio-Rad).
8.3.2. Load the rest of the samples (approximate 15ul) prepared in step 8.1 into the gel.
8.3.3. Use Precision Plus protein Kaleidoscope as the molecular marker (Bio-Rad)
8.3.4. Proceed to fix the gel with Gel fixation Solution (40% Methanol, 10% Acetic Acid) for
30 minutes.
8.3.5. Wash the gel with water for 10 minutes after fixing. Repeat twice.
8.3.6. Transfer the gel onto a filter paper.
8.3.7. Dry the gel using Bio-Rad Gel dryer (80OC constant temperature for 20 minutes for one
gel).
8.3.8. Ensure the gel is completely dry. Remove the gel from the dryer.
8.3.9. Spot 0.1ul of radioactive feed buffer mixed with blue loading buffer upon each band of
the protein marker. Allow 5-minute to air dry.
8.3.10. Ensure the gel is completely dry since any moisture will damage the phosphor screen.
Place the dry gel into the phosphor screen cartridge assembly. Allow to expose
overnight.
8.3.11. Image the exposed phosphor screen in Typhoon using the phosphor screen setting with
minimum resolution of 100μm.
8.3.12. Analyze the gel and compare the size of the protein made with the expected size.
9. References:
9.1. The manuals for the equipment are contained in the following server folderR:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU Manuals
9.2. E-Gel® Technical Guide. General information and protocols for using E-Gel® pre-cast agarose
gels. Version J July 10, 2007 25-0645
9.3. PureLink™ 96 PCR Purification Kit For rapid, high-throughput purification of PCR Catalog no.
K3100-96 Version B 21 April 2004 25-0716
9.4. Expressway™ Cell-Free E. coli Expression System Cell-free protein synthesis system for
expression of recombinant protein Catalog nos. K9901-00, K9900-96, K9900-97 Version A 15
February 2006 25-0890
9.5. QIAquick® Spin Handbook For QIAquick Gel Extraction Kit, November 2006.
9.6. Specific Activity is calculated using the following website:
http://www.ehs.uiuc.edu/rss/toolscalcs/raddecay.aspx
10. Calculations and formulas
10.1. Determine quantity of protein produced.
10.1.1. pmoles of protein:
= ((CPM*Total volume)/CPM Volume))*(1/2130000000000)*(1/Specific
Activity)*(1/1000)*(1/# of Met)*(75/0.003)*(1012)
10.1.2. μg of Protein:
= ((CPM*Total volume)/CPM Volume))*(1/2130000000000)*(1/Specific
Activity)*(1/1000)*(1/# of Met)*(75/0.003)*(Molecular Weight)*(1000000)
10.2. DNA concentration in mg/ml using absorbance at 260nm. C= A260*ε-1* l-1 with ε=40
ml*mg-1*mm-1and l=10 mm
11. Glossary
11.1.
11.2.
11.3.
11.4.
11.5.
11.6.
11.7.
11.8.
11.9.
11.10.
11.11.
11.12.
11.13.
11.14.
11.15.
11.16.
11.17.
11.18.
11.19.
11.20.
11.21.
11.22.
11.23.
USAMRIID United States Army Medical Research Institute for Infectious Disease
LVS live attenuated vaccine
FTU Francisella tularensis
IVT Coupled in vitro Transcription and Translation
LEE Linear Expression Element
PCR polymerase Chain Reaction
UV Ultra Violet Light.
dNTP deoxyribonucleotide triphosphate
TCA Tri Chloro Acetic Acid
MES 2-(N-morpholino) ethanesulfonic acid.
PBS Phosphate Buffered Saline
GEMS Genetic Experimental Management System.
DNA Deoxyribonucleic Acid
RNA Ribonucleic acid
E.coli Escherichia coli
TRIS trishydroxymethylaminomethane
EDTA ethylene diamine tetraacetic acid
TBE Tris Boric acid EDTA
CPM counts per minute
BSA Bovine Serum Albumin
Trx Thioredoxin
MAb Monoclonal Antibody
g is g force
12. Appendices
Primers
T7-Thio-Forward: GCGAAATTAATACGACTCACTATAGGG
Thio Reverse: CAATCCGCTTCCGCCTATGGCCAGGTTAGCGTCGAGG
5’-Univ-Forward: ATAGGCGGAAGCGGATTG
T7-Term-His-Univ-Fforward:
ACCCAACCTCCCTCCCACCATCATCATCATCATTAATAAAAGGGCG
T7-Term-Reverse: ATCCGGATATAGTTCCTCCTTTCAG